Cell detection based on protein array using modified glass slides. cells in a complex mixture, effectively immobilizing and specifically detecting Her2+ cells at a concentration of 102 SK-BR-3 cells/mL in 4 106 white blood cells/mL. Patients with a variety of cancers can have circulating tumor cell counts of between 1 and 103 cells/mL in whole blood, well within the range of this technology. Graphical abstract INTRODUCTION Protein microarrays have been used in many biomedical applications including the detection of proteins in serum, the analysis of proteinCprotein interactions, and the study of posttranslational modifications.1 Specifically, antibody-based proteomics can identify and validate cancer biomarkers as well as provide a diagnostic approach for identification of different tumor types.2,3 Recently, antibody microarrays have been used to identify metastatic breast malignancy as well as distinguish patients with pancreatic cancer from healthy controls.4,5 Additionally, because antibody microarrays also have the ability to capture cells, they allow the possibility of detecting rare cells such as circulating tumor cells (CTCs).6,7 Although there are numerous applications for antibody arrays, construction of these protein arrays is a significantly greater challenge compared with conventional DNA microarrays. The generation of antibody microarrays requires immobilization of the antibody on either hydrophobic or Nedocromil chemically reactive (e.g., epoxy, aldehyde, maleimide) surfaces.8C10 However, this approach can cause denaturation and loss of activity due to immobilization of the protein in a nonproductive orientation or nonspecific binding of the protein to the surface. Approaches to preserve the protein conformation include three-dimensional matrixes such as hydrogels and polyacrylamide, and light-directed biotinCavidin arrays.11,12 Alternatively, one can immobilize antibodies on a DNA array by first modifying the protein of interest with a single-stranded oligonucleotide.13,14 In general, this approach prevents protein denaturation and loss of binding activity associated Mouse monoclonal to BNP with printing antibodies on a solid support and potentially allows for greater control of the orientation of the surface bound antibodies.13C17 Not only do these arrays allow for facile and rapid generation of antibody arrays, they have also been shown to have superior binding characteristics when compared to standard antibody arrays. Utilizing DNA directed antibody immobilization on a DNA microarray also allows for concomitant detection of multiple biomolecules, biomarkers, genes or cell types on a single platform. The most common method for conjugating DNA to antibodies is usually by modification of surface uncovered lysine residues. However, coupling to the lysine residues results in a heterogeneous mixture of products which can interrupt antigen binding and cause the antibodies to aggregate.18C20 Random conjugation also prevents control of antibody orientation on the surface, which can lead to loss of activity and specificity. Peluso et al. reported up to a 10-fold Nedocromil increase in analyte binding capacity between a specifically oriented and a randomly oriented antibody using streptavidin-coated surfaces.11 In the context of immuno-PCR, there was a significant difference in signal when comparing Nedocromil site-specific and random DNA conjugation.21 Additionally, site-specific DNACFab conjugation has recently been used to develop an extremely sensitive homogeneous immunoassay, detecting PSA at concentrations of 0.27 ng/mL.22 The availability of genetically encoded unnatural amino acids with Nedocromil unique chemical reactivity can provide a solution to these challenges. Previously, we have site-specifically incorporated in good yields ( 2 mg/L shake flasks, 400 mg/L fermentation), purified by Protein G, and characterized by SDS-PAGE gel and electrospray-ionization mass spectrometry (ESI-MS) (Expected 47 860 Da; Observed 47 861 Da). As depicted in Physique 2A, lane 2, only one band is usually observed after Protein G purification, indicating 95% purity. The antibodyColigonucleotide conjugates were then produced via the protocol layed out in Kazane et al., utilizing an aminoxy-functionalized single-stranded oligonucleotide to achieve bio-orthogonal condensation with the ketone moiety and form a stable oxime linkage.21 Anti-Her2 S202pAcF Fab was conjugated to aminooxy-modified oligonucleotide sequences (C and D, Table S1) (100 M Fab, 3 mM oligonucleotide, 100 mM methoxy aniline, pH 4.5, 37 C, 16 h), purified by anion exchange chromatography (Mono Q 5/50 GL), and analyzed by SDS-PAGE (Determine 2A and S1). As depicted in Physique 2A, there is only one band (lanes 3 and 4) which correspond to the homogeneous site-specific Fab-oligonucleotide conjugate. Open in a separate window Physique 2 (A) SDS-page gel analysis of anti-Her2 S202pAcF Fab after conjugation to oligonucleotide-C and oligonucleotide-D. Conjugates were purified by.
Cell detection based on protein array using modified glass slides
